Preparation method and application of an enrichment material for extracellular vesicles

Abstract

The application belongs to the field of extracellular vesicle enrichment materials, and relates to a preparation method and application of an extracellular vesicle enrichment material. Polystyrene (PS) nanospheres are used as templates to in-situ grow ZIF-8 in ordered cavities by a multi-phase nucleation method induced by double solvents, to form a single crystal SOM-ZIF-8 material with a directional and ordered macroscopic microporous structure, and to make the whole material have an amino active functional group by exchanging 3-amino-1,2,4-triazole with 2-methyl imidazole ligands in the SOM-ZIF-8 material. Finally, SOM-ZIF-8-NH2 material is acylated with a phospholipid probe to obtain SOM-ZIF-8-phospholipid probe material. The prepared SOM-ZIF-8-phospholipid probe material can efficiently and quickly enrich EVs from a biological sample. In addition, biological analysis of proteins and nucleic acids in EVs by combining molecular biology experiments and micro ribonucleic acid sequencing (miRNA-seq) and other technologies can find potential cancer biomarkers, which provides a reliable method for downstream functional analysis of EVs and is conducive to in-depth study of the biological function of EVs in a biological sample.

Description

Technical Field

[0001] This invention belongs to the field of extracellular vesicle enrichment materials, and particularly relates to a method for preparing and applying an extracellular vesicle enrichment material. Background Technology

[0002] EVs are nanoparticles with a phospholipid bilayer structure secreted by cells. EVs play a crucial role in biological processes such as mediating intercellular communication, regulating immune responses, tumorigenesis, and metastasis. The demand for EV research has greatly promoted the development of EV isolation methods. Currently, most methods for isolating EVs from biological samples are based on principles such as ultracentrifugation, immunoaffinity, polymer coprecipitation, and size exclusion. However, these methods all have unavoidable limitations, such as being time-consuming, inefficient, cumbersome, and having low purity. Therefore, there is an urgent need to develop an effective and simple method for EV isolation. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention provides a method for preparing and applying an extracellular vesicle (EV) enrichment material. ZIF-8 is formed by in-situ crystallization on a monolithic polystyrene nanosphere template. Based on the ordered macro-microporous structure of single-crystal SOM-ZIF-8 material, a SOM-ZIF-8-NH2 material with amino groups on its surface is designed, and its surface-active functional groups are used to modify a phospholipid probe. The prepared SOM-ZIF-8-phospholipid probe material can rapidly and efficiently enrich EVs from biological samples such as cell culture media and plasma. Molecular biology experiments are then used to extract and analyze the nucleic acids and proteins contained within the EVs, and potential cancer biomarkers are screened.

[0004] The technical solution provided by this invention is as follows:

[0005] This invention provides a method for preparing an enrichment material for extracellular vesicles, comprising the following steps:

[0006] S1, prepare monodisperse polystyrene microspheres and ZIF-8 precursor solution; the ZIF-8 precursor solution is a methanol solution of zinc nitrate hexahydrate and 2-methylimidazole;

[0007] S2, monodisperse polystyrene microspheres are immersed in a precursor solution and dried to obtain an impregnated composite solid material;

[0008] S3. After treating the composite solid material with methanol and ammonia, it was degassed under vacuum, allowed to stand, dried, and then the monodisperse polystyrene microspheres were dissolved with an organic solvent to obtain the SOM-ZIF-8 solid material.

[0009] S4, SOM-ZIF-8 solid material, methanol and 3-amino-1,2,4-triazole are mixed and heated and stirred to obtain SOM-ZIF-8-NH2 material;

[0010] S5, a phospholipid probe with PEG-COOH at the end, SOM-ZIF-8-NH2 material, ethanol aqueous solution, Tris-HCl buffer, N-hydroxysuccinimide and carbodiimide are mixed and reacted to obtain SOM-ZIF-8-phospholipid material as the extracellular vesicle enrichment material.

[0011] Furthermore, the method for preparing the monodisperse polystyrene microspheres includes dispersing styrene and polypyrrolidone in an aqueous solution, adding potassium sulfate, and obtaining polystyrene microspheres through a water bath reaction; the preparation process is always protected by nitrogen gas; the mass ratio of styrene, polypyrrolidone, potassium sulfate and water is 13g:2.5g:0.2g:110g; the temperature of the water bath reaction is 60~90℃.

[0012] Furthermore, the particle size range of the monodisperse polystyrene microspheres is 300~400 nm.

[0013] Furthermore, in step 3, the volume ratio of methanol to ammonia is 1:1; the organic solvent is N,N-dimethylformamide; and the etching time is 48~72 h.

[0014] Furthermore, the phospholipid probe is selected from one of cholesterol, distearylphosphatidylethanolamine (DSPE), 1,2-dioleoyl-SN-glycerol-3-phosphoethanolamine (DOPE), dipalmitoylphosphatidylethanolamine (DPPE), and membrane-penetrating peptides.

[0015] Furthermore, the phospholipid probe is distearate phosphatidylethanolamine (DSPE), and the mass ratio of DSPE-PEG-COOH to SOM-ZIF-8-NH2 material is 1~10:1.

[0016] Furthermore, the mass ratio of DSPE-PEG-COOH to SOM-ZIF-8-NH2 material is 10:1.

[0017] Furthermore, the size of the SOM-ZIF-8 phospholipid material is 2~3 μm.

[0018] The present invention also provides an extracellular vesicle enrichment material prepared according to the above-described method for preparing an extracellular vesicle enrichment material.

[0019] The present invention also provides an application of the extracellular vesicle enrichment material described above in the enrichment of extracellular vesicles, comprising the following steps:

[0020] The biological sample, PBS solution, nonylphenol polyoxyethylene ether, and Triton X-100 were mixed to obtain a mixed solution;

[0021] The extracellular vesicle enrichment material described above is added to the obtained mixed solution, incubated, and then impurities are removed to complete the enrichment of extracellular vesicles.

[0022] Beneficial effects

[0023] The extracellular vesicle enrichment material, namely SOM-ZIF-8-phospholipid probe material, prepared in this invention is first formed by in-situ crystallization of ZIF-8 on a polystyrene (PS) nanosphere template. A single-crystal SOM-ZIF-8 material with an ordered macroporous microporous structure is then formed using a dual-solvent induction process. Based on the SOM-ZIF-8 material, 3-amino-1,2,4-triazole (Atz) is exchanged with the 2-methylimidazolium ligand in the SOM-ZIF-8 material to impart amino active functional groups to the entire material. Finally, an acylation reaction is carried out between the SOM-ZIF-8-NH2 material and the phospholipid probe to obtain the SOM-ZIF-8-phospholipid probe material. The prepared SOM-ZIF-8-phospholipid probe material can efficiently and rapidly enrich EVs from cell culture medium and plasma. Furthermore, by combining molecular biology experiments with microRNA sequencing (miRNA-seq) and other techniques to perform biological analysis of proteins and nucleic acids within EVs, potential cancer biomarkers can be discovered. This provides a reliable method for downstream functional analysis of EVs and facilitates in-depth research into the biological functions of EVs in biological samples.

[0024] The SOM-ZIF-8 material was mainly prepared by using PS spheres as templates and inducing ZIF-8 to crystallize in situ using a dual-solvent approach. The template was then dissolved with organic reagents to transform the material from a solid to a hollow structure, resulting in an ordered macroporous framework SOM-ZIF-8 material. Based on this, subsequent amination and phospholipid probe molecule modification were performed to finally obtain the SOM-ZIF-8-phospholipid probe material. The prepared material exhibits good physical and chemical stability, can be dispersed in PBS buffer for extended periods, and can be stored at 4 °C.

[0025] The enrichment principle of EVs by the SOM-ZIF-8 phospholipid probe material prepared by this method is based on the Zn content in the SOM-ZIF-8 framework material. 2+The SOM-ZIF-8 phospholipid probe material achieves synergistic bifunctional enrichment through two forces: metal chelation with the phosphate groups of the EV membrane and non-covalent enrichment achieved by the lipid tail insertion of surface-modified phospholipid probe molecules into the phospholipid bilayer. The interconnected macroporous framework structure maximizes the exposure of active sites, facilitating contact between EVs and these sites.

[0026] The material prepared by this method can efficiently enrich EVs in cell culture medium and plasma at room temperature. Compared with SOM-ZIF-8 material, the SOM-ZIF-8-phospholipid probe material modified with phospholipid probe molecules has a higher capture efficiency; and within 10 min, 1 mg of SOM-ZIF-8-phospholipid probe material can completely enrich EVs in 300 μL of cell culture medium, with a recovery rate of up to 80%. The captured EVs can also be used for downstream biological analysis. Attached Figure Description

[0027] Figure 1 Electron micrograph of SOM-ZIF-8-DSPE material.

[0028] Figure 2 Comparative analysis of the enrichment efficiency of SOM-ZIF-8 material and SOM-ZIF-DSPE material modified with different proportions of DSPE on EVs in cell culture medium, and WB images of EV marker proteins CD9 and TSG101.

[0029] Figure 3 Scanning electron microscope (SEM) and laser confocal microscope (DFM) images of SOM-ZIF-8-DSPE material before and after capturing EVs.

[0030] Figure 4 A graph showing the mRNA expression level analysis in EVs enriched with SOM-ZIF-8-DSPE material.

[0031] Figure 5 Differential gene volcano plot of miRNA-seq of EVs enriched in plasma using SOM-ZIF-8-DSPE material.

[0032] Figure 6 This is a comparison of the efficiency of SOM-ZIF-8-DSPE material with other traditional capture methods. Detailed Implementation

[0033] This invention uses monodisperse polystyrene microspheres (PS) as a template. After adding a ZIF-8 precursor, the precursor crystallizes in situ on the template using a methanol-ammonia solution to form a polyhedral structure. The internal microsphere template is then dissolved to prepare a hollow framework SOM-ZIF-8 material with a macroporous structure. The SOM-ZIF-8 framework material has a nanometer-scale wall thickness, which is beneficial for modifying phospholipid probe molecules onto its framework to obtain SOM-ZIF-8-phospholipid probe materials. The macroporous structure in the SOM-ZIF-8 material can provide a larger capture space for EVs.

[0034] This invention provides a method for preparing an enrichment material for extracellular vesicles, comprising the following steps:

[0035] S1, prepare monodisperse polystyrene microspheres and ZIF-8 precursor solution; the ZIF-8 precursor solution is a methanol solution of zinc nitrate hexahydrate and 2-methylimidazole;

[0036] S2, monodisperse polystyrene microspheres are immersed in a precursor solution and dried to obtain an impregnated composite solid material;

[0037] S3. After treating the composite solid material with methanol and ammonia, it was degassed under vacuum, allowed to stand, dried, and then the monodisperse polystyrene microspheres were dissolved with an organic solvent to obtain the SOM-ZIF-8 solid material.

[0038] S4, SOM-ZIF-8 solid material, methanol and 3-amino-1,2,4-triazole are mixed and heated and stirred to obtain SOM-ZIF-8-NH2 material;

[0039] S5, a phospholipid probe with PEG-COOH at the end, SOM-ZIF-8-NH2 material, ethanol aqueous solution, Tris-HCl buffer, N-hydroxysuccinimide and carbodiimide are mixed and reacted to obtain SOM-ZIF-8-phospholipid material as the extracellular vesicle enrichment material.

[0040] The specific preparation process is as follows:

[0041] 1) Add styrene monomer, polyvinylpyrrolidone, and deionized water to the reaction vessel;

[0042] 2) The above reaction system was magnetically stirred in an oil bath at 60~90℃ for 30 min under nitrogen protection;

[0043] 3) Add potassium sulfate to step 2) to initiate the polymerization reaction, and react in an oil bath at 60-90℃ for 24 h;

[0044] 4) The prepared colloidal monodisperse polystyrene (PS) product was transferred to a centrifuge tube and centrifuged to obtain PS microspheres;

[0045] 5) Prepare a ZIF-8 precursor solution composed of 2-methylimidazole and zinc nitrate hexahydrate, then immerse the PS microspheres in the ZIF-8 precursor, let it stand at room temperature for 1 h and then degas it under vacuum to ensure that the precursor is fully wetted into the PS template gaps.

[0046] 6) After removing the precursor solution, the PS template from step 5) is dried at 50 °C for 12 h to obtain the impregnated composite solid material;

[0047] 7) The solid material from step 6) was immersed in a methanol-ammonia solution, degassed under vacuum for 3 min, and then allowed to stand at room temperature and atmospheric pressure for 24 h before drying to obtain SOM-ZIF-8@PS material;

[0048] 8) Immerse the SOM-ZIF-8@PS material from step 7) in N,N-dimethylformamide to dissolve the PS spheres and obtain the SOM-ZIF-8 solid material;

[0049] 9) Add the SOM-ZIF-8 solid material and methanol solution from step 8) to the reaction vessel and disperse them ultrasonically;

[0050] 10) Add 3-amino-1,2,4-triazole ATZ to the reaction vessel of step 9);

[0051] 11) Place the above reaction system in a 50 °C water bath and stir for 1 h;

[0052] 12) The white precipitate collected by centrifugation is the SOM-ZIF-8-NH2 material;

[0053] 13) Add an ethanol solution containing a phospholipid probe with PEG-COOH at the end to a centrifuge tube, and simultaneously add a Tris-HCl buffer containing N-hydroxysuccinimide and carbodiimide;

[0054] 14) Add the SOM-ZIF-8-NH2 material from step 12) to the reaction vessel from step 13), and react for 6 h under uniform suspension.

[0055] 15) Wash the precipitate obtained in step 14) three times with deionized water and then dry it to obtain uniformly dispersed SOM-ZIF-8-phospholipid probe material.

[0056] In this embodiment, the mass ratio of styrene, polypyrrolidone, potassium sulfate and water is 13g:2.5g:0.2g:110g.

[0057] Specifically, in this embodiment of the invention, a PS nanosphere template is prepared by emulsification polymerization. The ZIF-8 precursor is induced to crystallize in situ on the ordered PS nanosphere template under the action of methanol and ammonia as dual solvents, forming SOM-ZIF-8@PS material. Then, the PS nanosphere template is dissolved and removed with N,N-dimethyldiamide, ultimately obtaining a single-crystal SOM-ZIF-8 material with a directional ordered macro-microporous structure. Furthermore, 3-amino-1,2,4-triazole (ATZ) is exchanged with the 2-methylimidazolium ligand in the SOM-ZIF-8 material to give the entire material amino-active functional groups. Finally, an acylation reaction is carried out on the SOM-ZIF-8-NH2 base with a phospholipid probe molecule with one end of HOOC-PEG, thus preparing the SOM-ZIF-8-phospholipid probe material.

[0058] SOM-ZIF-8 phospholipid material is a ZIF-8 material with a three-dimensional ordered macroporous framework structure. This ordered macroporous framework, with a size of approximately 2-3 μm, facilitates long-distance material transport. Simultaneously, the hollow wall structure promotes contact between the material and the active sites, maximizing the exposure and function of these sites. Therefore, it has played a significant role in many fields in recent years, including gas storage, photocatalysis, metal adsorption, and battery energy storage. For the enrichment of EVs, this framework material is rich in Zn. 2+ Chelation sites, stable properties, and macroporous structure. Therefore, EVs can enter the interior of the material through the macroporous structure and chelate with the framework material, thereby achieving EV separation.

[0059] In this embodiment, the particle size range of the monodisperse polystyrene microspheres is 100~600nm.

[0060] In this embodiment, the volume ratio of methanol to ammonia is 1:1; the dissolution time is 48~72h.

[0061] In this embodiment, the phospholipid probe is selected from one of cholesterol, distearylphosphatidylethanolamine (DSPE), 1,2-dioleoyl-SN-glycerol-3-phosphoethanolamine (DOPE), dipalmitoylphosphatidylethanolamine (DPPE), and transmembrane peptides. Specifically, phospholipid probes are often used for labeling and capturing EVs because their hydrophobic tail molecules can anchor and insert into the phospholipid bilayer of EVs.

[0062] In this embodiment, the phospholipid probe is distearate phosphatidylethanolamine (DSPE), and the mass ratio of DSPE-PEG-COOH to SOM-ZIF-8-NH2 material is 1~10:1.

[0063] Preferably, the mass ratio of DSPE-PEG-COOH to SOM-ZIF-8-NH2 material is 10:1.

[0064] Example 1

[0065] This invention provides a method for preparing an enrichment material for extracellular vesicles, comprising the following steps:

[0066] 1) Take 16 mL of styrene and remove the stabilizer successively with 5 mL of 10% sodium hydroxide and 5 mL of deionized water;

[0067] 2) Add 13 mL of styrene and 100 mL of an aqueous solution containing 2.5 g of polyvinylpyrrolidone to a 250 mL three-necked flask;

[0068] 3) The above reaction system was magnetically stirred in a 65 °C oil bath under vacuum for 30 min;

[0069] 4) Add 10 mL of an aqueous solution containing 0.2 g potassium sulfate to step 3) to initiate the polymerization reaction, and let the system react in an oil bath at 65 °C for 24 h;

[0070] 5) The prepared colloidal PS product was transferred to a centrifuge tube, centrifuged (3500 rpm, 12 h), and then dried at 60 ℃ for 12 h to obtain PS ball templates;

[0071] 6) Dissolve 8.15 g of zinc nitrate hexahydrate and 6.75 g of 2-methylimidazole in 45 mL of methanol solution to prepare ZIF-8 precursor solution. Then immerse the PS template in ZIF-8 precursor solution, let it stand at room temperature for 1 h, and then degas it under vacuum for 10 min to ensure that the precursor fully wets the gaps in the PS template.

[0072] 7) After removing the precursor solution, the PS template from step 6) is dried at 50 °C for 12 h to obtain the impregnated composite solid material;

[0073] 8) The solid material from step 7) was immersed in a methanol-ammonia solution (1:1, v / v), degassed under vacuum for 3 min, and then allowed to stand at room temperature and atmospheric pressure for 24 h before drying to obtain SOM-ZIF-8@PS material;

[0074] 9) The SOM-ZIF-8@PS solid from step 8) was immersed in N,N-dimethylformamide for 72 h to dissolve the PS spheres and finally obtain the SOM-ZIF-8 solid material;

[0075] 10) Take 50 mg of the SOM-ZIF-8 material from step 9) and ultrasonically disperse it in 40 mL of methanol solution;

[0076] 11) Dissolve 146.25 mg of ATZ in 10 mL of methanol;

[0077] 12) Add the reagents from steps 10) and 11) to a single-necked flask, place it in a 50 °C water bath, and stir to react for 1 h;

[0078] 13) After the reaction is complete, the product is centrifuged (3000 rpm, 5 min), the white precipitate is washed three times with methanol and then dried. The white product obtained is the SOM-ZIF-8-NH2 material.

[0079] 14) Add 1 mL of DSPE-PEG containing 10 mg to a 10 ml centrifuge tube. 2000 The acylation reaction system consisted of a 30% ethanol solution of -COOH, 4 mL of Tris-HCl buffer (pH=6.5) containing 50 mg of N-hydroxysuccinimide, and 114 μL of carbodiimide reagent.

[0080] 15) Add 10 mg of the SOM-ZIF-8-NH2 material from step 13) to step 14) and suspend at room temperature for 6 h.

[0081] 16) After the reaction is complete, take the reaction products in portions and transfer them to centrifuge tubes. Centrifuge (3000 rpm, 5 min) and collect the white precipitate.

[0082] 17) Wash the precipitate obtained in step 16) twice with PBS buffer to obtain SOM-ZIF-8-DSPE material. Add 1 mL of PBS solution to prepare a PBS solution of SOM-ZIF-8-DSPE with a concentration of 10 mg / mL for later use.

[0083] Example 2

[0084] Everything else is the same as in Example 1, except for DSPE-PEG. 2000 The mass ratio of -COOH to SOM-ZIF-8-NH2 material is 2:1.

[0085] Example 3

[0086] Everything else is the same as in Example 1, except for DSPE-PEG. 2000 The mass ratio of -COOH to SOM-ZIF-8-NH2 material is 5:1.

[0087] Example 4

[0088] Everything else is the same as in Example 1, except for DSPE-PEG. 2000 The mass ratio of -COOH to SOM-ZIF-8-NH2 material is 10:1.

[0089] Example 5

[0090] The present invention also provides an extracellular vesicle enrichment material prepared according to the preparation method of the extracellular vesicle enrichment material described in Example 1.

[0091] Example 6

[0092] The present invention also provides an application of the extracellular vesicle enrichment material according to Example 1 in the enrichment of extracellular vesicles, comprising the following steps:

[0093] 1) Add 50 μL of SOM-ZIF-8-DSPE material from step 17), 300 μL of cell culture medium, 100 μL of 0.1% nonylphenol polyoxyethylene ether / Tricon X-100 PBS solution and 550 μL of PBS buffer to a 1.5 mL EP tube.

[0094] 2) Incubate the mixture from step 18) at room temperature for 1 h;

[0095] 3) Centrifuge the system from step 19) (5000×g, 5min), discard the supernatant, and keep the bottom precipitate;

[0096] 4) Wash the precipitate from step 20) once with 500 μL of 0.01% nonylphenol polyoxyethylene ether / Troton X-100 PBS solution, and then wash twice with PBS to complete the enrichment of EVs.

[0097] Example 7

[0098] The present invention also provides an application of the extracellular vesicle enrichment material according to Example 1 in the enrichment of extracellular vesicles, comprising the following steps:

[0099] 1) Add 100 μL of the SOM-ZIF-8-DSPE material from step 17), 1000 μL of clinical plasma sample, and 123 μL of 0.1% nonylphenol polyoxyethylene ether / Tricon X-100 PBS solution to a 1.5 mL EP tube.

[0100] 2) Incubate the mixture from step 18) at room temperature for 1 h;

[0101] 3) Centrifuge the system from step 19) (5000×g, 5min), discard the supernatant, and keep the bottom precipitate;

[0102] 4) Wash the precipitate from step 20) once with 500 μL of 0.01% nonylphenol polyoxyethylene ether / Troton X-100 PBS solution, and then wash twice with PBS to complete the enrichment of EVs.

[0103] Effect evaluation

[0104] The PS microspheres prepared in Example 1 and the SOM-ZIF-8-DSPE material were characterized by electron microscopy as follows: Figure 1 As shown. Figure 1 (A) is a transmission electron microscope image of PS microspheres. The image shows that the PS microspheres are regular spherical with uniform particle size and a size of about 340 nm. Figure 1 (B) is a scanning electron microscope image of regularly stacked PS microspheres; Figure 1 (C) and Figure 1 (D) are transmission electron microscope (TEM) and scanning electron microscope (SEM) images of the modified SOM-ZIF-8-DSPE material, respectively. The prepared material has a hollow framework structure of about 2-3 μm and regular large pores on the surface, which is consistent with the expected results.

[0105] Figure 2 The capture efficiency of EVs modified with different proportions of DSPE molecules in Examples 1-4 was compared. EV marker proteins (CD9 and TSG101) were detected by Western blotting, and the gray levels of the protein bands were compared.

[0106] The results show that, compared with the SOM-ZIF-8 material, DSPE molecular modification significantly improves the capture efficiency of EVs, and when DSPE-PEG... 2000 The optimal DSPE molecular modification ratio was achieved when the mass ratio of -COOH to SOM-ZIF-8-NH2 material was 10:1, demonstrating that the prepared SOM-ZIF-8-DSPE material effectively enriches EVs through bifunctional synergistic enrichment.

[0107] Figure 3 Scanning electron microscope (SEM) and laser confocal microscope (DFM) images of SOM-ZIF-8-DSPE material before and after capturing EVs.

[0108] Figure 3 (AB) are scanning electron microscope (SEM) images before and after EV capture. Compared to before capture, a large number of EVs are enriched in the macropores of the material surface after capture. Meanwhile, Figure 3 (C) After fluorescently labeling EVs, they were co-incubated with the material, and further observation under a confocal microscope revealed that the material had a high recovery rate of EVs.

[0109] Figure 4 This is a graph showing the mRNA expression level analysis in EVs enriched with SOM-ZIF-8-DSPE material.

[0110] Figure 4 (A) is Example 6, which shows that EVs were captured from the culture medium of four types of colorectal cancer (CRC) cells and the total amount of RNA extracted from 1 mL of sample was approximately 2000-3000 ng. Figure 4(B) shows that EVs in the four CRC cell cultures can be detected with varying degrees of cancer-related mRNA expression, demonstrating that the EVs captured by this method can be used for downstream bioanalysis.

[0111] Figure 5 Differential gene volcano plot of miRNA-seq of EVs enriched in plasma using SOM-ZIF-8-DSPE material.

[0112] Example 7 uses plasma as a sample to capture EVs. Figure 5 After capturing extracellular endothelial cells (EVs) from plasma samples collected from clinical CRC patients, differentially expressed genes were obtained by sequencing the miRNAs within the EVs. Differentially regulated genes were detected in both in situ CRC samples (T) and metastatic CRC samples (M), and most of these differentially expressed genes were closely related to the occurrence, development, and metastasis of CRC, consistent with previous literature reports. This demonstrates that this method is applicable to the enrichment of EVs from clinical plasma samples and can serve as a novel approach for discovering potential cancer biomarkers.

[0113] Figure 6 This is a comparison chart of the efficiency of SOM-ZIF-8-DSPE material with other traditional capture methods.

[0114] The SOM-ZIF-8-DSPE method captures far more EVs than the ultracentrifugation (UC) method. In terms of capture time, the SOM-ZIF-8-DSPE method only takes 1 hour, which is 1 / 8 of the time required by the polymer coprecipitation method (Precipitation). This indicates that the method has a higher capture efficiency than the traditional method.

[0115] From the examples and Figure 1-6 It is evident that the SOM-ZIF-8-DSPE material prepared in this manner can achieve highly efficient enrichment of EVs in cell culture medium and plasma through the synergistic effect of metal chelation and DSPE molecular anchoring, with a capture efficiency superior to traditional capture methods. Analysis of the expression levels of its contained mRNA, miRNA, and protein using PCR and Western blotting experiments, along with miRNA sequencing technology for EV miRNA analysis, can help screen for potential CRC-related biomarkers, thereby maximizing the clinical diagnostic value of EVs.

Claims

1. A method for preparing an extracellular vesicle enrichment material, characterized in that, Includes the following steps: S1, prepare monodisperse polystyrene microspheres and ZIF-8 precursor solution; the ZIF-8 precursor solution is a methanol solution of zinc nitrate hexahydrate and 2-methylimidazole; S2, monodisperse polystyrene microspheres are immersed in a precursor solution and dried to obtain an impregnated composite solid material; S3, after treating the composite solid material with methanol and ammonia, vacuum degassing, standing, and drying, the monodisperse polystyrene microspheres are then dissolved in an organic solvent to obtain the SOM-ZIF-8 solid material; the volume ratio of methanol to ammonia is 1:1; the organic solvent is N,N-dimethylformamide; and the dissolution time is 48~72h. S4, SOM-ZIF-8 solid material, methanol and 3-amino-1,2,4-triazole are mixed and heated and stirred to obtain SOM-ZIF-8-NH2 material; S5, a phospholipid probe with PEG-COOH at the end, SOM-ZIF-8-NH2 material, ethanol aqueous solution, Tris-HCl buffer, N-hydroxysuccinimide and carbodiimide are mixed and reacted to obtain SOM-ZIF-8-phospholipid material as the extracellular vesicle enrichment material. The method for preparing the monodisperse polystyrene microspheres includes dispersing styrene and polypyrrolidone in an aqueous solution, adding potassium sulfate, and obtaining polystyrene microspheres through a water bath reaction; the preparation process is always protected by nitrogen gas; the mass ratio of styrene, polypyrrolidone, potassium sulfate and water is 13g:2.5g:0.2g:110g; the temperature of the water bath reaction is 60~90℃; The particle size range of the monodisperse polystyrene microspheres is 100~600nm; The phospholipid probe is distearate phosphatidylethanolamine (DSPE), and the mass ratio of DSPE-PEG-COOH to SOM-ZIF-8-NH2 material is 10:

1. The size of the SOM-ZIF-8 phospholipid material is 2~3 μm.

2. An extracellular vesicle enrichment material prepared by the method for preparing extracellular vesicle enrichment material according to claim 1.

3. The application of the extracellular vesicle enrichment material according to claim 2 in the enrichment of extracellular vesicles, characterized in that, Includes the following steps: The biological sample, PBS solution, nonylphenol polyoxyethylene ether, and Triton X-100 were mixed to obtain a mixed solution; The extracellular vesicle enrichment material described in claim 2 is added to the obtained mixed solution, incubated, and then impurities are removed to complete the enrichment of extracellular vesicles.

Images